Fig. 4: Functional characterization of AgmF as an unusual dehydratase catalyzing a reversible reaction.
From: Efficient biosynthesis of nucleoside cytokinin angustmycin A containing an unusual sugar system
a HPLC analysis (λ = 254 nm) of AgmF reaction with 2 as substrate. Std, the authentic standards of 2 and 1; AgmF, AgmF reaction with 2 as substrate; -AgmF, reaction using 2 as substrate but without AgmF added; -(2), AgmF reaction without 2 added. b HPLC analysis (λ = 254 nm) of AgmF-catalyzed reversible reaction with 1 as substrate. Std, the authentic standards of 2 and 1; AgmF, AgmF reaction with 1 as substrate; -AgmF, reaction using 1 as substrate but without AgmF added; -(1), AgmF reaction without 1 added. c Homology structure model of AgmF. This structure is constructed on the basis of Rv3248c from Mycobacterium tuberculosis H37Rv (PDB 3CE6), and the active 2 and NAD+ binding sites are indicated in the rectangular region. The sites marked out are proposed to be essential for the binding of 2. d HPLC analysis (λ = 254 nm) of the reactions of AgmF and its variants. Std, the authentic standards of 2 and 1. AgmF, reaction with the intact AgmF added; lines labeled H56A, D129A, K184A, D188A, and H299A show the individual reactions of AgmF variants; -(2), AgmF reaction without substrate 2 added. e Proposed enzymatic mechanism for the AgmF-catalyzed reaction.
